Current methods for intracellular protein analysis mostly require the separation of specific organelles or changes to the intracellular environment. However, the functions of proteins are determined by their native microenvironment as they usually form complexes with ions, nucleic acids, and other proteins. Here, we show a method for in situ cross-linking and analysis of mitochondrial proteins in living cells. By using the poly(lactic-co-glycolic acid) (PLGA) nanoparticles functionalized with dimethyldioctadecylammonium bromide (DDAB) to deliver protein cross-linkers into mitochondria, we subsequently analyze the cross-linked proteins using mass spectrometry. With this method, we identify a total of 74 pairs of protein-protein interactions that do not exist in the STRING database. Interestingly, our data on mitochondrial respiratory chain proteins ( ~ 94%) are also consistent with the experimental or predicted structural analysis of these proteins. Thus, we provide a promising technology platform for in situ defining protein analysis in cellular organelles under their native microenvironment.

目前的细胞内蛋白质分析方法大多需要分离特定的细胞器或改变细胞内环境。然而，蛋白质的功能是由其天然微环境决定的，因为它们通常与离子、核酸和其他蛋白质形成复合物。在这里，我们展示了一种原位交联和分析活细胞中线粒体蛋白的方法。通过使用用二甲基双十八烷基溴化铵 (DDAB) 功能化的聚乳酸-乙醇酸 (PLGA) 纳米颗粒将蛋白质交联剂递送到线粒体中，我们随后使用质谱法分析交联的蛋白质。通过这种方法，我们总共鉴定了 STRING 数据库中不存在的 74 对蛋白质-蛋白质相互作用。有趣的是，我们关于线粒体呼吸链蛋白（约 94%）的数据也与这些蛋白的实验或预测结构分析一致。因此，我们提供了一个有前景的技术平台，用于在细胞器的天然微环境下进行原位定义蛋白质分析。